Cathepsin D Expression Levels in Nongynecological Solid Tumors: Clinical and Therapeutic Implications

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Review Cathepsin D expression levels in nongynecological solid tumors: Clinical and therapeutic implications

Gaetano Leto, Francesca M. Tumminello, Marilena Crescimanno, Carla Flandina & Nicola Gebbia Section of Chemotherapy, Department of Oncology, Policlinico Universitario ‘P. Giaccone’, Palermo, Italy

Received 19 September 2003; accepted in revised form 13 January 2004

Key words: cathepsin D, lysosomal proteinases, matrix-metalloproteinases, metastasis, neoplasm, plasminogen activator, prognosis, tumor progression

Abstract Cathepsin D is a lysosomal acid proteinase which is involved in the malignant progression of breast cancer and other gynecological tumors. Clinical investigations have shown that in breast cancer patients cathepsin D overexpression was significantly correlated with a shorter free-time disease and overall survival, whereas in patients with ovarian or endometrial cancer this phenomenon was associated with tumor aggressiveness and a degree of chemoresistance to various antitumor drugs such as anthracyclines, cis-platinum and vinca alkaloids. Therefore, a lot of research has been undertaken to evaluate the role and the prognostic value of cathepsin D also in other solid neoplasms. However, conflicting results have been generated from these studies. The discrepancies in these results may, in part, be explained with the different methodological approaches used in order to determine the levels of expression of the enzyme in tumor tissues and body fluids. Further investigations using well-standardized techniques may better define the clinical significance of cathepsin D expression in solid tumors. Nevertheless, evidence emerging from these studies indicates that this proteinase seems to facilitate early phases of tumor progression such as cell proliferation and local dissemination. These findings support the concept that cathepsin D may be a useful marker for identifying patients with highly malignant tumor phenotypes who may need more aggressive clinical treatment; this enzyme may also be considered as a potential target for a novel therapeutic approach in the treatment of solid neoplasms.

Abbreviations: cAMP – cyclic adenosine monophosphate; BCC – basal cell carcinoma; CB – cathepsin B; CD – cathepsin D; CL – cathepsin L; CNS – central nervous system; CRC – colorectal cancer; EGFR – epidermal growth factor receptor; GC – gastric cancer; HCC – hepatocellular carcinoma; H&N – head and neck; HIF-1 – hypoxia–inducibile factor 1; IGF – insulin-like growth factor; IGFBP – insulin-like growth factor binding protein; LSCC – laryngeal squamous cell carcinoma; MMP-2 – matrix-metalloproteinase-2; MMP-9 – matrix-metalloproteinase-9; NSCLC – non small cell lung cancer; PCNA – nuclear proliferating antigen; PSA – prostatic speciﬁc antigen; SCC – squamous cell carcinoma; SCLC – small cell lung cancer; TGF-β – transforming growth factor beta; TSH – thyroid stimulating hormone; uPA – urokinase-type plasminogen activator

Introduction been located in chromosome 11p15 and contains 9 exons [18]. Mice deficient in this gene present alterations in the Structure and biological functions of cathepsin D small intestine (i.e., necrosis associated with thrombosis of small vessels) and a large-scale destruction of lymphoid cells Cathepsin D (CD) (EC 3.4.23.5) is a lysosomal acid pro- in the spleen and timus [17]. These findings further confirm teinase which, in the range of pH 2.8–5.0, can degrade the active role of CD in the renewal, remodelling and in ap- structural and functional proteins, peptides, peptide precurs- optosis of a wide variety of tissues [1, 5–8, 11, 12, 17]. This ors and hormones [1–4]. However, it has been shown that enzyme is synthetized as an inactive 52kDa precursor form the biological functions of this enzyme are not only con- [19]. Its activation is processed intracellularly by sequential fined to the metabolic degradation of intracellular proteins proteolytic cleavage which first involve the removal of the but also comprise some other important biological processes 44-amino-acid propeptide. This cleavage yelds an active 48- (Table 1) [1–17]. In humans, the gene coding for CD has kDa single chain molecule which is then cleaved by cysteine Correspondence to: Gaetano Leto, Sezione di Chemioterapia, Diparti- proteinases into two active chain forms of 34 kDa (heavy mento di Oncologia, Policlinico Universitario ‘P. Giaccone’, Via del Vespro chain) and 14 kDa (light chain) [19]. CD is ubiquitously 129, 90127 Palermo, Italy. Tel: +39-091-6552617; Fax: +39-91-6552760; present in animal and human tissue, with qualitative and E-mail: [email protected] quantitative differences in its distribution among different 92 G. Leto et al. organs or cell types of the same organ [1, 4, 11, 12, 14– 24, 58–62]. This hypothesis is currently supported by several 16, 20–23]. This uneven distribution seems to be related to clinical observations which have shown that, in the human the different biological function carried out by this enzyme brain, altered CD levels can be frequently associated with in tissue [1]. these diseases [21–23, 57–62]. The mechanism(s) by which CD may promote the progression of CNS tumors are still Cathepsin D and cancer unknown. However, certain in vitro studies showed that anti- CD has also been shown to be widely distributed also in CD antibodies, in a dose-dependent fashion, significantly human tumors. However, many of these tumors present reduced the invasive potential of human glioblastoma cells. altered processing, secretion and activity levels of this en- These findings indicated that this proteinase may facilitate zyme as compared to normal tissues [4, 22–30]. These the adhesion and the subsequent invasion of these tumor phenomena, which may also be associated with the aggress- cells to the extracellular matrix of host tissue [63, 64]. The ive behavior of tumors, suggest that CD, in concert with potential role of CD in the modulation of the invasive activity other proteolytic enzymes involved in the metastatic proof CNS tumors has been further supported by some im- cess such as cysteine proteinases cathepsins B (CB) and L munohistochemical investigations which demonstrated that (CL), serine-proteinase urokinase-type plasminogen activ- the invading cells of astrocytomas, glioblastomas, oligo- ator (uPA) and matrix-metalloproteinases-2 (MMP-2) and dendrogliomas and mixed gliomas expressed CD, MMP-2 -9 (MMP-9), may promote the malignant progression of and MMP-9, and that the switching to an invasive pheno- neoplastic diseases [25–36]. The mechanism(s) by which type of these tumors was followed by an increase in CD CD may facilitate this process has still not been fully elu- expression levels [64]. More recently, Castino et al. [65] cidated. However, in vitro studies carried out mainly on have hypothesized that this proteinase, in association with human breast or ovarian cancer cell lines evidenced that this CB, can modulate some proteolytic occurrences of caspase- proteinase may stimulate tumor cell proliferation, invasion dependent apoptosis which are essential for neuroblastoma and metastasis by various mechanism(s) summarized in Fig- cell survival. This may, in part, explain the cytotoxic effect ure 1 (reviewed by Rochefort et al. in [19, 26, 27]). As a induced by pepstatin A, a naturally occurring inhibitor of consequence, several studies have been undertaken in order CD, on neuroblastoma cells after a 72-h incubation period to evaluate its clinical significance in breast cancer and in [65, 66]. These findings further showed that CD may act at other gynecological neoplasms. These studies showed that, different steps of the growth of CNS neoplasms and sugges- in node-negative breast cancer patients, CD overexpression ted a potential clinical role of this proteinase as an indicator was associated with an increased risk of recurrence and of aggressiveness and prognosis of these tumors. However, death [26, 37, 38]. These findings were not confirmed in little research has been undertaken so far to test this hypo- male breast cancer, which seems to be biologically differ- thesis. Some biochemical studies have shown, at least in ent from female breast tumor [39, 40]. Conflicting results human gliomas, a close relationship between up-regulation were also obtained in other gynecological malignancies [42– of this enzyme and malignant progression of this tumor [63]. 54]. However, these investigations additionally highlighted a Recently, Castilla et al. [62] reported a significant associ- close association between altered expression levels of CD ation between an increased immunocytochemical expression and the degree of aggressiveness and chemoresistance of of CD in meningiomas and recurrence, whereas no correla- ovarian or endometrial tumors [42–55]. In this context, sev- tion with patients’ outcome was noted. These observations eral investigations have also been undertaken to assess the indicate that the evaluation of CD expression may be use- clinical significance of CD expression in other nongynecolo- ful in identifying more aggressive forms of CNS neoplasms, gical solid tumors. Extensive updated reports on the results and may be seen as an attractive target for innovative thera- of these studies are still lacking in literature. This review peutic approaches to these tumors [63–65]. However, due article summarizes these data and discusses their clinical to the lack of extensive clinical studies, its prognostic value implications. remains to be determined.

Cathepsin D in central nervous system tumors Cathepsin D in head and neck tumors Immunohistochemical and immunoenzymatic studies have CD is commonly found in animal and human central nervous system (CNS) tissue [1, 20, 22–24]. Although its biological shown that CD expression levels may be found to be altered role in CNS is still not well deﬁned, experimental evidence in different tumors of the head and neck (H&N) including those of the oral cavity, parotid gland, salivary glands, suggests that this proteinase, along with other proteolytic enzymes of the apoptotic process, namely caspases [56], oro- and hypo-pharinx and larynx [67–73]. The biological signiﬁcance of such alterations has still not been properly may be involved in the regulation of neuronal cell death, understood. However, there is a lot of experimental and clin- survival and differentiation [57]. Therefore, altered expression levels of this enzyme may result in detrimental effects ical evidence to suggest that this phenomenon may be related to the dissemination of H&N neoplasms, and ultimately to on the biological and physiological functions of neuronal cells which may lead to severe degenerative disorders of the onset of more aggressive forms of these tumors [68– CNS such as Alzheimer’s disease or CNS neoplasms [22, 75]. This latter observation is further supported by recent in vitro studies which have shown that, in human laryngeal Cathepsin D expression in solid tumors 93

Table 1. Main biological functions in which cathepsin D appears to be involved.

Reference number

Metabolic degradation of intracellular proteins [1] Processing, activation and degradation of polypeptide hormones, growth factors and receptors [1–4] Biological regulation of programmed cell death [5–10] Tissue remodelling and renewal [1, 11, 12, 17] Activation of latent precursor forms of other proteolytic enzymes [13] Activation of neutrophils and leukocytes [14, 15] Monocyte-mediated ﬁbrinolysis [16]

Figure 1. Step 1. Cathepsin D may promote tumor cell proliferation: a) by acting as an autocrine mitogen through the interaction with a transmembrane receptors (?) [19, 26, 34, 35, 140, 141]; b) by activating growth factors [19, 26, 34, 35]; c) by degrading growth factors inhibitors [36]; d) by interacting with growth factor receptors [19, 26, 27, 138–141]; e) by affecting the apoptotic process [5, 34, 36, 57, 65, 113–115]. Step 2. Cathepsin D may directly degrade extracellular matrix (ECM) and facilitate tumor cell invasion [19, 25, 26, 63]: 2a) this phenomenon may additionally induce the release of biologically active forms of growth factors preincorporated in the ECM which, in turn, may stimulate tumor cells to proliferate [19, 26]. Step 3. Cathepsin D may trigger off a proteolytic cascade, by activating latent precursor forms of other proteolytic enzymes and chemiotactic factors which may facilitate ∗ ECM degradation, invasion, angiogenesis and metastasis formation [13, 24-26, 35, 102, 116, 127, 161, 170]. MMPs – matrix-metalloproteinases; uPA – urokinase-type plasminogen activator; ECM – extracellular matrix carcinoma (LSSC) cell lines, increased intracellular levels hypothesis that CD may play a role in the early phases of of CD and uPA, were associated with the onset of resistance H&N cancer progression has been further corroborated by to different classes of antitumor agents such as doxorubi- some of our recent clinical observations, which showed that, cin, cis-platinum and vincristine [76]. The mechanisms by unlike in early clinical stage (I/II) LSCC, CD levels were which CD may induce the progression H&N tumors remain not increased in locally advanced tumors (stage III/V) as to be clarified. However, clinical evidences suggest that CD compared to normal adjacent mucosa [67, 77]. In addition, may well aid the early phases in this process by facilitating our data also highlighted significantly higher uPA levels in the proliferation, adhesion and local dissemination of tumor tumor tissues when compared to its normal counterpart [77]. cells. In fact, immunohistochemical studies by Goussa et A comparative analysis of the distribution of CD and uPA al. [72] on 39 tissue samples from invasive LSCC showed in stage III/IV LSCC showed that CD was more noticeably a significant correlation between the expression levels of expressed only in tumors with a high proliferation rate, as CD and the hyaluronic acid receptor CD44 whereas, in 97 compared to those with a low proliferation index, whereas patients with oral cancer, Vigneswaran et al. [73] observed uPA was more noticeably expressed in node-positive tu- a significant association between increased CD expression mors than in node-negative tumors, in stage-IV tumors than and proliferation rate, histological grade and presence of stage-III tumors and in aneuploid multiclonal tumors than in metastasis. Moreover, Kawasaki et al. [74] recently demon- aneuploid-monoclonal or diploid tumors. [77]. These results strated in 78 patients with oral squamous cell carcinoma further indicated that, at least in the case of LSCC pro- (SCC), a close relationship between the CD expression and gression, CD seems more likely to be involved in tumor the pattern of invasion, clinical stage, nodal status, nuclear cell proliferation and local growth whereas uPA seemed to proliferating antigen (PCNA) and shorter survival time. The be implicated in subsequent events in this process such as 94 G. Leto et al. the invasion and further dissemination of tumor cells. Al- authors speculated that this phenomenon was probably the though these findings might suggest a prognostic role for result of metabolic disturbances in the catabolic degradation CD in LSCC, conflicting results have emerged from studies of thyreoglobulin in tumor tissue. Further immunoenzymatic aimed at evaluating this hypothesis. Immunohistochemical investigations by Métayé et al. [83] carried out on a small investigations by Resnick et al. [78] on 88 LSCC patients number of patients (n = 12) showed that CD content did not find any significant relationship between CD ex- levels were significantly higher in thyroid carcinoma, toxic pression, degree of nodal involvement, disease-free survival adenoma or Graves’ disease, than in normal tissue or be- or overall survival time. Similar studies by Seiwerth et al. nign nodules. However, no difference was observed between [79] in 61 untreated LSCC patients, in part, confirmed these thyroid carcinoma and Graves’ disease or toxic adenoma. In- findings. However, these authors reported a significant cor- terestingly, these studies and later investigations of the same relation between CD immunoreactivity in both epithelial and group carried out on 34 patients evidenced a significant asso- stromal cells and patient survival. On the other hand, Lazaris ciation between CD levels in primary tumors and tumor size et al. [80] observed by immunocytochemistry that, among 64 [83, 87]. Additional observations of these authors showed LSCC patients, those with CD positive lymph nodes were that the activity levels of this enzyme in different human at higher risk of relapse. The positive relationship between thyroid tissues were 3.0, 2.3 and 1.3 times higher in cancer CD expression and nodal involvement was further confirmed tissue or toxic adenoma, in Graves’disease, and in cold be- by the immunoradiometric studies of Maurizi et al. [81] on nign nodule respectively, than in normal thyroid tissue [4]. In 63 patients with primary LSCC. In addition these authors addition, in a small number of samples, it was also noted that showed that, at the cut-off level considered (13.8 pmol/mg mean CD activity levels were higher in anaplastic carcino- of protein), CD tumor content significantly correlated also mas than in well differentiated thyroid carcinomas [4]. These with metastasis-free survival and overall survival. However, findings indicated the existence of a possible correlation other immunoradiometric or immunoenzymatic studies, in- between degree of CD expression and aggressive behavior cluding our own investigations, failed to find any correlation of thyroid tumors, and suggested that CD could play a role between CD tumor levels and the biological and clinical in their propensity to metastasize. This hypothesis was sup- parameters of progression or patients’ survival [77, 82]. The ported by some studies by Ruhoy et al. [88], who showed discrepancies in these results may be, in part, explained by that CD immunostaining was higher in follicular carcino- the different assay methods and/or antibody used and/or by mas than in follicular adenomas and that this phenomenon the cut-off limits considered. However, it also cannot be was greatest in extensively invasive follicular carcinomas. ruled out that the different criteria of selection, the num- These observations suggested that this proteinase could be ber of patients, the different anatomical site considered and of clinical interest as a prognostic marker in thyroid tumors the different follow-up periods may also account for these [83, 88]. To date, however, no extensive clinical studies to conflicting results. Although the prognostic significance of assess this hypothesis have yet been undertaken. A single CD remains controversial, evidence emerging from these study on 44 patients with medullary carcinoma reported studies suggests that this proteinase may be useful as as a a weak correlation between the immunohistochemical ex- biological marker for identifying patients with primary tu- pression of CD and poor prognosis [89]. Thus, additional mor at increased risk of recurrence and onset of resistence clinical investigations to establish the prognostic signific- to therapeutic treatment. Further studies with standardized ance of this enzyme are needed. However, the results of the techniques may better define the clinical role of CD in H&N existing studies indicated that CD may be useful as a specific cancer. marker to identify tumors endowed with a greater invasive and metastatic potential. This findings may greatly improve the therapeutic approach and the prognosis of these tumors. Cathepsin D in thyroid tumors

In the human thyroid gland CD is one of the proteolytic en- Cathepsin D in lung cancer zymes involved in the metabolism of thyreoglobulin [3, 4]. In some pathological conditions, such as Graves’ disease and The role of CD and its clinical significance in lung cancer is toxic adenoma, increased activity or content levels of this still controversial. Some biochemical and immunoenzymatic proteinase have been shown to be associated with a hyper- studies have questioned its direct involvement in the growth functional thyroid [4, 83]. This phenomenon appears to be and advance of at least of some histological types of lung induced by TSH as these pathological conditions are related cancer. For instance, Ledakis et al. [90] reported that, in to a permanently stimulated cAMP transduction pathway non-small-cell lung cancer (NSCLC), CD activity or content which mediates the effects of TSH on thyroid cells including levels, had not increased, unlike CB and CL, as compared the synthesis and secretion of CD [4, 84, 85]. Therefore, a with adjacent normal lung tissue. Moreover, other immuno- number of investigations have also been carried out to as- histochemical studies by Fontanini et al. [91] undertaken on sess the clinical significance of CD expression in thyroid 108 NSCLC patients showed that, CD expression levels was tumors. Early biochemical studies by Sinadinovic et al. [86] associated with smaller size (< 3 cm), less advanced tumors reported that the patients’ papillary thyroid carcinoma tis- (T1), more differentated (G1-2) tumors and non-squamous sue presented an enhanced proteolytic activity of lysosomal histotypes. In addition, in non-squamous histotypes, CD im- acid proteases as compared to normal thyroid tissue. The munoreactivity was associated with early staged (S1) and Cathepsin D expression in solid tumors 95 lymph node-negative (N0) tumors whereas no correlation some immunohistochemical studies have described a differ- was observed with proliferation indices such as DNA ploidy, ential immunostaining pattern of CD expression in primary S-phase fraction, PCNA or Ki-67. On the contrary, studies gastric carcinoma tissue [96]. The most intense staining was on patients with small cell lung cancer (SCLC) evidenced noted in tumor cells at the advancing margin of the tumor. that a low staining for CD or its absence was associated with This peculiar localization was significantly correlated with a prolonged survival [92]. Moreover, Higashiyama et al. certain clinical parameters of GC progression, such as the [93] observed that, in patients with stage I lung adenocar- clinical stage and occurrence of lymph-node metastasis [96]. cinoma, the subcellular localization of CD in the basal or Other biochemical and immunohistochemical studies further infranuclear side of the cytoplasm in tumor cells, as well as confirmed the relationship between CD activity and the in- its presence in stromal cells within the tumor tissues, was vasive potential of GC [97, 98]. Therefore, several clinical associated with a worse prognosis. These results sugges- investigations were carried out to evaluate the clinical signi- ted that CD may play a differential role in the regulation ficance of this proteinase, mainly in patients with curatively of the growth and differentiation of various histological resected cancer, that was at risk of recurrence. Immunohisto- types of lung cancer. As a consequence, its prognostic im- chemical studies by Allgayer et al. [99] on 203 consecutive pact in these neoplasms might be strongly affected by the patients showed that CD expression levels were significantly histological type of the tumor and/or its status in stromal associated with overall survival and a shorter disease-free in- cells. Therefore, the clinical significance of CD expression terval. Multivariate analysis identified CD as an independent in different histotypes of lung cancer needs to be better parameter for disease-free interval [99]. In contrast, some defined through precisely conceived studies and standard- of our immunoenzymatic studies undertaken on 57 patients ized immunohistochemical methods. Furthermore, studies with operable GC showed, that CD levels in GC tissue, have been recently undertaken to evaluate the clinical utility unlike CB and CL, did not increase as compared to paired of serum-CD activity levels in the therapeutic monitoring of normal gastric mucosa nor did they correlate with some of lung cancer patients [94]. These investigations, carried out the clinicobiological parameters of progression of this tu- on 21 patients with stage-II/III SCLC, showed that the activ- mor including the patients’ outcome [100]. It is likely that ity levels of this enzyme were significanty higher in SCLC the different methods and antibodies used, and/or the differ- patients than in healthy subjects. These levels markedly de- ent number of patients investigated may account for these clined after surgery albeit remained three times higher than conflicting results. On the other hand, some recent immuno- those determined in the control group [94]. These observa- histochemical studies by Goishi et al. [101] showed that, in tions suggested that serum CD activity may be potentially 136 patients with tumors invading the submucosa and mus- useful for the therapeutic monitoring of lung cancer patients cularis propria, CD expression correlated significantly with undergoing surgical and/or clinical treatments. However, ex- the increasing incidence of lymph-node metastasis. These tensive clinical studies with a larger number of patients are observations were in agreement with those of Ikeguchi et al. needed to assess this hypothesis. [102] who showed, in 160 patients with early GC, a significant correlation between CD expression and occurrence of micro-lymph-node metastasis. Further observation by the Cathepsin D in tumors of the gastrointestinal tract same group of a larger number of patients (n = 478) evidenced that the percentage of CD-positive cancer cells was Gastric cancer higher in diffuse-type carcinoma than in intestinal-type carcinoma [103]. These findings were also confirmed by the Immunohistochemical analysis of CD distribution in gastric immunoenzymatic studies of Garcia et al. [104]. Further- tissues has shown that this proteinase is widely present in more, Ikeguchi et al. [103] reported that, in both diffuse different cell types of normal gastric mucosa, as well as in and intestinal type of carcinoma, CD expression levels were benign and malignant gastric diseases [21, 95–103]. How- associated with depth of tumor invasion and a worse disease- ever, its specific role in the malignant progression of gastric specific five-year survival rate whereas CD levels in stromal cancer (GC) has still not been clarified. Early immunocyto- cells were associated with either depth of invasion or with chemical studies did not evidence any significant difference a worse five-year survival rate only in the intestinal type in the distribution of this proteinase between normal mucosa carcinoma [103]. These findings indicated that the CD of and inflammatory gastric diseases [95]. Moreover, these stromal cells seems to play an active role in the invasion of studies showed the absence of any immunoreactivity to CD this type of tumor and suggested that it may strongly influ- in intestinal metaplasia or dysplasia or in well differenti- ence the prognostic significance of this enzyme. Although ated gastric adenocarcinoma. However, a strong and diffuse these studies did not fully clarify the mechanisms by which staining for CD was observed in poorly differentiated ad- CD may induce the growth and spread of GC, they provided enocarcinomas and in signet ring cell carcinoma [95]. These evidence for the clinical utility of CD as a marker of lymph findings suggested a correlation between CD expression node involvement and indicate that this proteinase may be levels and degree of differentiation of gastric cancer but did a good candidate as prognostic parameter for predicting the not clarify whether this enzyme might have a specific role in clinical outcome of GC patients. the progression of this tumor. However, clinical observation supports the hypothesis that CD may play a key role in this process, probably, by facilitating tumor cell invasion. In fact, 96 G. Leto et al.

Colorectal cancer normal tissue [107]. However, unlike the described enzyme activity, a significant correlation was observed between CD Several investigations have shown that CD levels may be tumor content and tumor size or grade [107]. These findings found to be altered in colorectal cancer (CRC) and that this further underline that different results may be obtained in phenomenon is frequently associated with CRC progression accordance to the methodological approaches and/or number (105–112). These data indicate that CD may have a role in of patients used. For instance immunohistochemical analysis the growth and spread of CRC. However, the mechanisms of CD expression carried out by Theodoropoulos et al. [108] by which CD may facilitate this process are not well un- on 60 surgical CRC samples showed that the presence of derstood. In vitro studies showed that HT-29 human colon CD immunoreactivity in stromal cells was associated with a carcinoma cell lines presented an altered secretion of CD more invasive phenotype, while Kanber et al. [109] showed which was associated with a less differentiated state of these that stromal CD expression was also related to tumor stage. cells [113]. This phenomenon, which appears to facilitate On the other hand, Arao et al. [110] reported that the immun- the degradation of the extracellular matrix by tumor cells, ostaining pattern of CD in tumor cells, but not the incidence seems to be related to altered levels of cell associated cer- of CD positive tumors, was associated significantly with amide, an important mediator of the apoptotic process [114, lymphatic invasion. Furthermore, Oh-e et al. [111], follow- 115]. As CD seems to be involved in the biological regu- ing the immunocytochemical evaluation of the intracellular lation of programmed cell death [5–10, 57, 65, 113–115] pattern of expression of CD in 254 invasive CRC, observed it is conceivable that its altered secretion may also result a significant correlation between this parameter or positive in disturbances of the normal apoptotic process which may expression of CD in stromal cells, and incidence of lymph- facilitate tumor cell growth. However, other experimental node metastasis. These findings indicate that, as previously observation showed that CD may induce the growth and described for GC, CD stromal cells appear to influence the spread of this tumor by other mechanisms such as the activ- invasive potential of CRC tumors. Therefore, analysis of ation of latent precursor forms of other proteinases involved CD expression in both tumor and stromal cells can be re- in the metastatic process. This latter hypothesis is supported garded as a useful predictor for lymph-node metastasis, and by some studies of van der Stappen et al. [13] who ob- consequently, may have a clinical relevance in predicting served that, in an in vitro model of CRC progression, the the clinical outcome of CRC patients. These observations conversion of non tumorigenic adenoma derived cell lines indicate that the immnunohistochemical evaluation of CD to a highly tumorigenic phenotype was associated with an expression is more reliable than other methods for investig- eight-fold increase of pro-CB and to an enhanced secretion ating the clinical significance of this proteinase in CRC. In of CD. Moreover, these studies also evidenced that the ac- conclusion, experimental and clinical findings indicate that tivation of CB was mediated by CD. This phenomenon may, CD appears to promote the progression of CRC, probably in turn, trigger off the proteolytic cascade which leads to by affecting the apoptotic process of tumor cells and/or by the degradation of extracellular matrix and the subsequent facilitating tumor cell invasion, and that this enzyme may invasion of host tissues by malignant cells (Figure 1). The be clinically relevant as a predictive marker of lymph-node potential involvement of CD in CRC invasion has been fur- involvement and poor clinical outcome in CRC patients. ther sustained by recent observation which has shown that, in human HCT116 colon carcinoma cells, the overexpres- Liver cancer sion of hypoxia-inducibile factor 1 (HIF-1) stimulates the expression of genes encoding for several factors, includ- Several experimental and clinical studies support the hypo- ing CD, which contribute to extracellular-matrix invasion thesis that CD may also be implicated in the onset and pro- [116]. These experimental findings suggest that CD may gression of liver tumors. For instance, in vitro and in vivo ob- have a potential clinical role as marker of aggressiveness servations evidenced that the extremely fast-growing Morris and poor prognosis in CRC patients. Several clinical stud- hepatoma 777 cells presented altered, intracellular pro- ies have been performed to assess this hypothesis. Our early cessing and increased secretion of a precursor form of CD investigations on 68 matched paired sets of CRC and normal whereas the ascitic fluid and the plasma of rats transplanted tissue sample homogenates showed significantly increased with Yoshida AH-130 hepatoma presented elevated levels of CD activity levels in tumor tissues as compared to adja- CD activity [118, 119]. Interestingly, increased CD activ- cent paired normal mucosa and it was demonstrated for ity levels were also observed in human hepatoma tissues the first time that this activity was significantly higher in as compared to its normal counterpart [120]. These experi- early clinical stage CRC (i.e., Dukes’ stage A) than in later mental observations were further confirmed by clinical find- clinical stages (i.e, Dukes’ B, C and D) [105]. These lat- ings, which showed that CD activity or content levels were ter observations, which further indicated an involvement of significantly elevated in sera of patients with chronic liver CD in the early stages of CRC growth and progression, diseases, such as active hepatitis, cirrhosis and/or hepato- were not confirmed by subsequent studies from other au- cellular carcinoma (HCC), as compared to healthy subjects thors [106]. These discrepant results might be due to the [121–124]. These findings suggest an active involvement of different number of patients considered. On the contrary, CD in the malignant progression of liver tumors. Experi- our later immunoenzymatic studies on 21 matched paired mental studies undertaken to investigate the mechanisms by CRC and normal mucosa samples did not evidence any sig- which CD may trigger this pathological process suggested nificant difference in CD content between tumor and paired that this proteolytic enzyme seems to facilitate the prolifera- Cathepsin D expression in solid tumors 97 tion and dissemination of hepatic tumor cells. In fact, some tried to assess the clinical significance of the serum levels in vivo studies in mice showed that intraperitoneal injections of CD, and also of CB and CL in patients with pancreatic of purified preparation of CD stimulated DNA synthesis and carcinoma or pancreatitis. Our results showed that in cancer mitosis in the intact liver of these mice [125, 126]. As CD patients, unlike observed for CB and CL, CD serum levels seems to promote in vitro tumor cell proliferation, it can be were lower than those measured in normal subjects nor were hypothesized that this proteinase may stimulate tumor cells the enzyme levels correlated with any of the biological and to proliferate through its mitogenic activity [19, 26]. How- clinical parameters of progression of this tumor [134]. These ever, experimental evidence to support this hypothesis has findings confirmed previous immunocytochemical observa- not yet been forthcoming. Furthermore, other in vitro studies tions by Nakata et al. [135], who demonstrated a lack of showed that human hepatoma cell line PLC/PRF/5 actively correlation between CD expression in pancreatic tumor cells secretes CD in addition to transforming growth factor β and the presence of metastatic foci in lymph nodes and in (TGF-β) and fibronectin [127]. The authors of these studies other organs. However, our data also showed that CD serum speculated that the secreted CD may activate latent forms of concentrations were significantly more elevated in patients TGF-β which, in turn, regulates the secretion of fibronectin, with acute or chronic pancreatitis as compared to healthy a strong inducer of chemiotaxis. These events may lead to subjects or cancer patients [134]. This phenomenon was a further migration and invasion of surrounding tissues by noted also for CB, but not for CL. These observations indic- tumor cells. However, in this case too, experimental and ate that CB and CL might well appear to be more relevant clinical evidences which supports this mechanism are still than CD as prognostic markers in pancreatic cancer. This lacking. The hypothesis that CD may play an active role in hypothesis was recently confirmed by immunohistochem- the onset and progression of liver tumors have led several ical studies by Niedergethmann et al. [136] who reported studies to assess its clinical significance in these tumors as a significant correlation between the expression levels of well as in some premalignant liver diseases such as cirrhosis. these proteinases and an unfavorable clinical outcome in pa- Our studies and those of Brouillet et al. [123, 124] showed tients with operable pancreatic cancer. These findings seem that, in patients with cirrhosis, CD antigen levels were sig- to rule out a direct role for CD in the progression of pan- nificantly higher than those determined in HCC patients. In creatic cancer. However, the different serum pattern of this addition, our data also showed that patients with steatosis proteinase in pancreatic cancer and pancreatitis might be a had significantly higher CD serum levels as compared to useful additional parameter in the differential diagnosis of healthy subjects, but these levels were significantly lower these diseases. than those measured in patients with liver cirrhosis and/or HCC [124]. These findings and other experimental data indicated that CD, in concert with other proteolytic enzymes, Cathepsin D in tumors of the genitourinary tract could be involved in the process of tissue remodelling which occurs during the evolution of cirrhosis [121, 128–130]. As Prostatic cancer this process may be associated with the malignant transformation of liver tissue, it can be speculated that CD may At present the role of CD in prostatic cancer is not well contribute also to the onset of malignant lesions in cirrhotic known. However, experimental evidence indicates that this tissue [123, 124, 131]. These observations suggest that CD proteinase may likely stimulate the growth of this tumor by may be potentially useful as a biochemical marker for identi- interacting with hormone receptors or growth factor recept- fying those patient with cirrhosis who risk developing HCC. ors and/or through its mitogenic activity. In fact, in vitro On the other hand its prognostic value in these tumors re- studies suggested that CD seems to facilitate the prolifera- mains to be assessed. Unfortunately, to date few clinical tion of prostatic tumor cells induced by insulin-like growth studies have been carried out toward this aim. A recent im- factor (IGF), by proteolytic degradation of IGF-binding pro- munohistochemical study on 85 HCC patients showed that teins (IGFB) or, in the case of androgen-dependent tumor CD expression, was correlated with the histological grade cells, by hydrolizing androgen receptors [137–139]. Inter- but the prognostic value of this enzyme to predict the clinical estingly, this latter phenomenon did not occur in normal outcome for these patients was not assessed [132]. prostatic tissue. On the other hand, Vétvicka´ et al. [140] showed that several human prostatic cancer cells secreted Pancreatic cancer an enzymatically inactive pro-CD, containing an activation peptide localized in the N-terminal amino-acid region 27– There are not very many studies into the role, and the clinical 44, which induces tumor cell proliferation and motility, significance, of CD in pancreatic cancer. In 1986 Yamaguchi probably, by interacting with an unknown transmembrane and Kawai [133] first reported that human pancreatic tumor receptor [140, 141]. This hypothesis was corroborated by cell line HPC-YT actively secreted in vitro a‘Cathepsin- some experimental in vivo observations which demonstrated D-like enzyme’ which was different from that present in that the administration of anti-27–44 peptide antibodies to normal pancreas. The authors suggested that this form might nude mice transplanted with LNCaP human prostatic can- be responsible for the degradation of the host extracellular cer cells or MDA-MB-231 breast cancer cells inhibited the matrix and that it might facilitate the invasion of this tu- growth of these tumors [140, 141]. However, Konno et al. mor. However, no further studies to confirm this hypothesis [142] showed that the antibiotic brefeldin A inhibited in vitro have been carried out. On the basis of these observations we 98 G. Leto et al.

Table 2. Clinical signiﬁcance of cathepsin D expression in nongynecological solid tumors.

Tumor Reference Assay methods No. of patients Correlation with Clinicopathologicalb Survivalc parameters

CNS Neuroblastomas 24 IHC 13 G NA Meningiomas 62 IHC 86 G DFS Gliomas 64 IHC 45 I NA

Head and neck

Different 69 IRMA 53 No NA anatomical sytes 70 IRMA 92 No NA 71 IRMA 111 G NA 75 IHC 34 N NA Salivary gland 68 IHC 44 Histological type NA Oral cavity 73 IHC 97 P, G, M NA 74 IHC 78 N,S,I,PCNA OS 75 IHC 34 N NA Laryngeal SCC 77 EIA 57 No No 78 IHC 88 No No 79 IHC 61 No OS 80 IHC 64 No DSF 81 IRMA 63 N DFS, OS

Thyroid

4 EA/IHC 107 G NA 83 IRMA 14 T NA 87 IRMA 32 T NA 88 IHC 34 G NA 89 IHC 44 S No

Lung

91 IHC 108(NSCLC) T,N,G,Sd NA 92 IHC 13 (SCLC) No OSd 93 IHC 152 (AdenoK, Stage I) Number of scars OS

Gastrointestinal tract

Oesophageal SCC 182 IHC 154 I, p53, Ki-67 No Gastric 95 IHC 21 G – 96 IHC 44 N, S NA 97 EA 42 I, N, G NA 98 IHC 29 + 15 adenomas I, N NA 99 IHC 203 G OS 100 EIA 57 No No 101 IHC 136 N NA 102 IHC 160 N No 103 IHC 478 I OS Colorectal 105 EA 68 Sd NA 106 EA 27 No NA 107 EIA 21 T, G NA 108 IHC 60 I, S OS 109 IHC 34 + 24 adenomas I NA 110 IHC 254 G, I, N NA 111 IHC 31 + 29 adenomas S NA Cathepsin D expression in solid tumors 99

Table 2. Continued.

Tumor Reference Assay methodsa No. of patients Correlation with Clinicopathologicalb Survivalc parameters

Liver 123 IRMA/ serum 27 No NA 124 EIA/ serum 56 No NA 132 IHC 85 G NA Pancreas 134 EIA serum 22 No NA 135 IHC 21 No No

Genitourinary tract

Prostate 146 EIA 20 No NA 147 IRMA 15 No NA 148 IHC 69 S NA 149 IHC 102 Gleason score NA 150 IHC 61 Gleason, ploidy NA 151 IHC 105 No No 152 IHC 71 No No 153 EIA/densitye 80 S, M No 154 EIA 72 I, S, M NA Bladder 155 IHC 77 Rb, CD44, Gd,Sd,P53d NA 156 IHC 105 G, Sd DFSd,OSd 157 IHC 60 G, Sd DFSd 158 IHC 177 G, I, EGRF, P53, S-phase OS 159 IHC 20 (T1) No No 160 IRMA 93 No No 161 IHC 32 No No 167 IHC/Western blot 23 I No

Melanoma and other skin tumors

Melanoma 169 IRMA 51 NA DFS 171 IHC 147 I DFS 172 EIA/plasma 108 Md No SCC, BCC, Bowen disease 173 IHC 46 I NA SCC 174 IHC 53 I, M DFS

aIHC – immunohistochemistry; IRMA – radioimmunoassay; EIA – enzyme immunoassay; EA – enzyme activity. bT – tumor size; G – tumor grade; N – nodal involvement; S – stage; M – metastasis; P – proliferation rate; I – depth of invasion. cDFS disease-free survival; OS – overall survival; NA – not assessed. dInversely correlated with cathepsin D expression. eCathepsin D density: ratio Cathepsin D serum content/prostate volume. the proliferation of LNCaP, PC-3 and DU-145 human pro- cycle regulatory retinoblastoma protein (pRB) and cyclin- static cancer cells. These effects, which were more marked dependent kinase inhibitor WAF1 (p21) respectively [142]. in LNCaP cells, were associated with an overexpression of These findings suggest that CD may have a differential role pro-CD induced by a blocking of maturation (i.e., activa- in modulating the growth of androgen-dependent and inde- tion) of CD caused by this antibiotic. These results seem to pendent prostatic cancer cells. This hypothesis is supported be in contrast with those reporting a stimulating effects of by recent in vitro studies which show that pro-CD secreted pro-CD on proliferation of breast or prostatic cancer cells. by androgen-independent PC-3 human prostatic carcinoma However, as LNCaP are androgen-dependent cancer cells, cell lines, following its conversion to pseudo-CD, could and brefeldin A has been shown to induce a dramatic re- generate angiostatin from plasminogen and that this phe- duction (> 90%) in the level of expression of androgen nomenon might prevent angiogenesis-dependent growth of receptors [143], it might conceivably be hypothesized that the metastasis [144]. However, evident proof that CD may the inhibiting effects of this antibiotic on cell growth are the modulate also in vivo the growth of prostatic cancer by these consequence of a down-regulation of these receptors which mechanisms is still lacking. On the other hand, clinical stud- may also mediate the proliferating effects of pro-CD. On the ies aimed at assessing the role and the prognostic impact of other hand, the inhibiting effects of brefeldin A on androgen- this enzyme in prostatic cancer have generated conflicting indipendent PC-3 and DU-145 prostatic tumor cells seemed results, according also to the method used. Immunoblot ana- to be induced by different mechanisms involving the cell- lysis by Cherry et al. [145] reported that human prostatic 100 G. Leto et al. cancer tissue expressed a mature form of CD, with a higher Bladder cancer catalytic activity, whereas normal or benign prostatic hyperplasia predominantly expressed an inactive precursor form The pattern of CD expression in normal and pathological of the enzyme. However, immunoenzymatic studies by Yang bladder tissue and its clinical significance in bladder can- et al. [146] which analyzed the distribution of CD in the cer has been extensively investigated mainly by immuno- cytosol fractions of 22 samples of hyperplastic tissue and 20 histochemical methods [21, 155, 156]. Early studies by of prostate cancer tissue, showed no difference in enzyme Dickinson et al. [156] on 105 samples of transitional blad- content between these tissues nor any correlation between der carcinoma showed that CD was expressed in 100% of CD tumor levels and other clinicobiological parameters such normal urothelium whereas only 51% of tumors were CD as degree of differentiation and expression of sex hormones positive. In addition, these studies evidenced a significant while other prognostic parameters were not considered. On inverse correlation between CD expression and tumor mor- the contrary, immunoradiometric studies by Chambon et al. phology, tumor stage or grade whereas no correlation with [147] on 15 human prostate cancer tissue samples showed DNA ploidy was observed. Univariate analysis showed that that cystosolic CD concentrations were more elevated in tu- negative staining for CD was associated with a poor pro- mor tissues than in normal prostate or prostatic hyperplasia. gnosis, while multivariate analysis failed to demonstrate However, when the authors further analyzed by immuno- any correlation between CD expression and overall sur- histochemistry, the distribution of CD in these tissues, they vival. These results tallied with other immunohistochemical observed, according to their score system, a higher expres- studies which reported an inverse correlation between CD sion of CD in benign prostatic hyperplasia [147]. On the score and tumor grade [157]. Furthermore, these studies other hand, other immunohistochemical studies by Makar showed that patients with a high PCNA labelling index and et al. [148] on 69 cases of primary adenocarcinoma of the CD-negative tumors had a significantly poorer prognosis prostate showed, according to the score method reported by compared to those with a low PCNA index and highly CD- these authors, a significant correlation with the pathological positive group while multivariate analysis indicated that both stage but not with the Gleason grade. Unlike these observa- these parameters were not independent prognostic factors tions, later studies by Maygarden et al. [149] showed in a [157]. Therefore, CD was considered a useful tool for identi- larger number of patients (n = 102) a significant correla- fying the malignant potential of bladder transitional cell tion between CD expression and Gleason’s combined score. carcinoma, and may provide additional information for pre- Furthermore, quantitative immunohistochemical studies by dicting survival when stratifying for tumor grade. In this Ross et al. [150] on 61 prostatic carcinoma biopsies further context, immunohistochemical studies by Lipponen [158] confirmed this latter finding. In addition, these authors also on 177 patients reported that the strong expression of CD demonstrated a significant correlation between CD expres- detected in 40% of bladder tumor specimens was associated sion and DNA-ploidy, but not with serum prostate specific with tumor grade 2–3, S-phase fraction, muscle invasive antigen (PSA) levels, pathological stage or post resection growth and overexpression of EGFR. These findings, in part, disease recurrence [151]. The failure of CD to the pre- clash with the previous ones. These conflicting data may dict clinical outcome for patients with clinically localized partially be explained by the different number of patients prostatic carcinoma was further supported by other immun- considered. Interestingly, this author also showed that CD ohistochemical studies [151, 152]. These findings suggest was expressed in macrophage-like cells at the invasion front that in prostatic cancer CD appeared to be of clinical rel- of the tumor which were infiltrated by inflammatory cells evance as an indicator of disease progression but not as a and tumor cells overexpressing EGFR or p53 protein. Mul- prognostic parameter. This hypothesis has been corroborated tivariate analysis showed that the presence of CD-positive by recent immunoenzymatic studies by Hara et al. [153] tissue macrophage, in addition to other variables, was an who have shown that serum CD and its density (i.e., ratio independent prognostic factor. These studies indicated that, CD serum levels/prostate volume) were significantly higher similarly as reported for other neoplasms, stromal CD, may in patients with metastatic disease as compared to those have a role in modulating the invasive activity of this tumor without metastasis. However, either CD serum levels, or its and may influence its prognostic significance. On the con- density did not significantly correlate with patients’ survival trary, Ozer et al. [159] did not find any prognostic value rate. Moreover, Miyake et al. [154] have recently observed of CD immunostaining in 20 patients with high-grade T1- that serum CD combined with systemic biopsy and/or PSA stage primary bladder cancer. Furthermore, imunoenzymatic levels may be useful as predictive marker of extraprostatic studies by Salman et al. [160] reported that CD content extension of the tumor in patients who have undergone rad- levels, detemined from 93 bladder tumor tissue samples, ical prostatectomy. These results indicated that CD may be did not correlate with the clinical parameters of progression an additional marker to identify patients with more aggress- considered or with prognosis. More recently, immunohisto- ive forms of prostatic cancer needing specific therapeutic chemical studies by Carrascosa et al. [161] on 32 patients treatment. However, the use of well standardized method- with invasive bladder carcinoma further confirmed these ob- ologies are needed to better assess the clinical role of CD in servations. These findings render the clinical role of CD the management of this tumor. in bladder cancer controversial. It appears likely the conflicting results are due to different methods and numbers of patients used in these studies. Moreover, as CD in stromal cells seems to modulate the invasive activity of this cancer Cathepsin D expression in solid tumors 101

[156, 158] this phenomenon should be taken into account in Cathepsin D as therapeutic target in cancer treatment evaluating the prognostic significance of this enzyme. These observations indicate that among the analytical methods The experimental and clinical findings suggesting a role for used to assess the clinical role of CD in bladder cancer im- CD in tumor progression, imply that the modulation of its munohistochemical methods which can separately assess the biological activity by the use of its specific inhibitors or an- prognostic impact of CD expression in tumor cells or stromal tibodies may have a clinical relevance in the treatment of cells appear to be the more reliable than other methods. solid neoplasms. Experimental in vitro and in vivo studies Further investigations with well standardized immunocyto- showed that anti-CD antibodies significantly reduced, in a chemical methods may better clarify the clinical role of CD dose-dependent fashion, the invasive potential human glio- in bladder carcinoma. blastoma cells, whereas antibodies raised against the 27–44 activation peptide of pro-CD administered to nude mice with human breast or prostatic tumors inhibited the growth of Cathepsin D in melanoma and other skin tumors these tumors [63, 140, 141]. However, further investigations to evaluate the potential clinical applications of these anti- There is a clear evidence that a number of intracellular bodies in cancer treatment have not yet been pursued. On and extracellular proteolytic enzymes may play a major the other hand, most experimental studies have been carried role in the onset and progression of melanoma and other out to evaluate the therapeutic activity of specific inhibitors skin tumors [162–165]. In fact several in vitro studies have of CD. These investigations have been mainly undertaken shown that human melanoma cells may release different pro- with pepstatin A, a naturally occurring inhibitor of CD and teinases, including CD, which may cooperate to degrade other aspartyl proteinases [1, 66]. In vitro studies by Castino the extracellular matrix, thus facilitating the invasion of this et al. [65] evidenced that this inhibitor, at 100 µM concen- tumor [31–33, 162, 166]. Immunohistochemical, immun- tration, was cytotoxic for some human neuroblastoma cell oenzymatic and biochemical studies have shown that CD lines. These authors hypothesized that the cytotoxic effects is markedly expressed either in vitro in human metastatic induced by pepstatin A may be the consequence of the in- melanoma cells or in vivo in primary and metastatic melan- hibition of CD activity which, in these tumor cells, seems oma tissue [166–171]. Interestingly, some of these invest- to modulate caspase-dependent apoptosis [9, 57, 65]. On igations evidenced, by immunohistochemistry and Western the other hand, our previous in vivo studies showed that the blot analysis, that CD was always present in dysplastic nevi intraperitoneal administration of pepstatin A induced a sig- but only in 18% of nevocellular nevi while it was absent in nificant reduction in the number of spontaneous lung or liver normal melanocytes [166, 167]. These observations were metastases in mice transplanted with Lewis Lung carcinoma, further indication that the presence of this enzyme seemed MCa mammary carcinoma or M5076 ovarian reticulum cell to be associated with melanoma development and progres- sarcoma but not in B16 melanoma or L1210-tumor bearing sion and that it might be of clinical interest as a prognostic mice [66, 175]. Interestingly, the administration of pepstatin marker. This hypothesis has been confirmed by a number A in combination with an antitumor agent with a broad of clinical investigations which have reported a significant spectrum of activity such as doxorubicin, to Lewis Lung or correlation between CD expression levels, determined by M5076-tumor-bearing mice, resulted in an additive effect on immunocytochemistry, in primary melanoma tissue and poor metastasis formation as compared to that induced by each clinical outcome [169–171]. On the contrary, other biochem- single agent [66]. The inhibiting effects of pepstatin A on ical studies have shown that CD plasma levels were not of metastasis formation did not seem to be due to the direct clinical value for identifying patients with malignant melan- cytotoxic activity of this agent on tumor cells, as the growth oma at high risk of recurrence. The correlation between of primary tumors in mice was not affected by its administra- CD expression and aggressive behavior of tumors has been tion. Moreover, our preliminary in vitro experiments showed observed also in other skin tumors. Immunohistochemical that this inhibitor, at concentrations of up to 1× 10−5 M, studies by Kawada et al. [173] showed that CD expression (i.e., 10 times lower than that tested by Castino et al. [65] increased in SCC, but not in patients with Bowen’s disease, on human neuroblastoma cells), induced in Lewis Lung or seborrhoic keratosis or basal cell carcinoma (BCC). More M5076 tumor cells, a marked inhibition of intracellular CD recently, Goldmann et al. [174], by analyzing the immun- activity, but no citotoxic effects. It could, thus, be specu- ostaining pattern of CD expression in 53 specimens from lated that the inhibition of CD activity may also account primary SCC of the skin, noted that CD and type-IV colla- for the therapeutic activity of pepstatin A. However, other genase were significantly overexpressed at the invading front unknown pharmacological effects induced by this inhibitor of metastasized tumors, as compared to those which were as well as the inhibition of other aspartyl proteinases which not. These findings suggested that CD, in concert with other may account for its antimetastatic activity cannot be ruled proteolytic enzymes, may be also involved in the SCC skin out. Nevertheless, these findings suggest a potential role of invasion. These results suggest that CD may be of value as pepstatin A and its analogs in the adjuvant therapy of solid biochemical marker to identify highly aggressive forms of tumors [25, 65, 66, 176]. However, more detailed inform- these tumors and therefore to identify high-risk patients for ation about the pharmacological and toxicological profile adjuvant therapy [162, 170, 171]. of these substances, as well as on their range of activity 102 G. Leto et al. is needed before assessing their potential clinical value in processes are still controversial, though recent findings in- cancer treatment. dicate that CD may modulate p53-mediated apoptosis and cell chemosensitivity to antitumor agents [182]. Clinical studies which have shown a significant correlation between Conclusions CD and P53 overexpression in different tumors, support this hypothesis [81, 117, 155, 158, 183]. These findings sug- Experimental and clinical observation indicate that CD may gest that CD may be also viewed as a potential attractive facilitate the growth and spread of solid tumors by acting target for the drawing-up of new therapeutic strategies in the at various phases of this process (Figure 1). Other evid- treatment of solid tumors, and in circumventing the onset of ence suggests that CD may also be involved in upstream antitumor drug-induced resistance. events of tumor progression such as carcinogenesis. This latter hypothesis is supported by some in vitro studies which reported that the intracellular expression levels of CD were Acknowledgements significantly altered during the oncogenic transformation of murine fibroblasts, or during the conversion of nontumori- The authors wish to thank Dr Paolo Mignatti, New York genic colon adenoma-derived cells to a highly tumorigenic University School of Medicine, for the helpful discussion phenotype [177, 178]. These results might also, in part, ac- and remarks on the manuscript and Michele Tumminello for count for the increased serum levels of this enzyme noted his editorial assistance. This work was supported, in part, by in certain pre-malignant conditions [121, 123, 124, 128, funds from Ministero della Istruzione, Università e Ricerca 129]. Therefore, the evaluation of CD expression levels in (MIUR) (ex quota 60%). tumor tissues, and/or body fluids, is proposed as prognostic parameter for predicting the clinical outcome for cancer patients [179]. Although, there is, to date, a general consensus References on the prognostic role of CD in female breast cancer [19, 26, 37, 38, 179], conflicting results have been obtained in 1. Barrett AJ. Cathepsin D and other carboxyl proteinases. In Barrett other gynecological tumors, as well as in other solid neo- AJ (ed): Proteinases in Mammalian Cells and Tissues. Amsterdam: Elsevier/North Holland Biomedical Press 1977; 209–29. plasms (Table 2). 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